The arrangement of these TFBS along the sequence of a ECR differs among zebrafish, mouse and human (Fig. 4), consistent with the lack of sequence conservation noted at these ECRs.
The mouse ECRs were cloned in the same zebrafish reporter vector used for the zebrafish constructs, and injected into zebrafish (the proportion of live fish showing expression driven by a ECR in a given tissue is shown in Additional file 2: Table S1).
Hence, on the basis of the similarity in their A/B domains when aligned with other insect EcR members, one cloned cDNA encodes an EcR-A isoform with a conserved 20 amino acid sequence (specific A-box) at the C-terminal of the A/B domain of all isoform-A receptors, and the other one is a SeEcR-B1 isoform.
During metamorphosis the nervous system is remodeled for adult function, the timing of which is coordinated by the steroid hormone 20-hydroxyecdysone (ecdysone) through the ecdysone receptor, a heterodimer of the nuclear receptors EcR (isoforms are EcR-A, EcR-B1, or EcR-B2) and Ultraspiracle (USP) (reviewed in [6]).
Three EcR isoforms (EcR-A, EcR-B1, and EcR-B2) [20] and one USP exist in the higher Diptera, such as Drosophila melanogaster [21].
Ever since the Drosophila EcR gene was cloned and the three EcR isoforms (EcR-A, EcR-B1 and EcR-B2) were characterized [21], two isoforms of EcR (EcR-A and EcR-B1) were only reported in a few lepidopteron species, including Bombyx mori, Choristoneura fumiferana and Chilo suppressalis [22], [23], [24].
Entire coding regions of Drosophila foxo, ecr-A, ecr-B1 and usp cDNAs were isolated by RT-PCR using cDNA made from w[1118] wandering larvae.
The gene ecr, encodes three receptor isoforms, EcR-A, EcR-B1 and EcR-B2, that form heterodimers with the Drosophila retinoid X receptor (RXR) homologue Ultraspiracle (USP) (Yao et al., 1993).
Dominant negative versions of the EcR-A, EcR-B1 and EcR-B2 isoforms have been reported to cause local and global phenotypes that were overlapping in some instances and seemingly specific in others.
For ecr-A, ecr-B1 and usp RT-PCR, forward primers were designed for gene specific sequences with a Flag-tag sequence on the 5′-end and reverse primers were designed for gene specific sequences including native stop codons.
Within that month 54 patients had a consultation with an ECR GP and all were asked to complete the questionnaire.
